The discovery, development and commercialization of the imidazolinone compounds has given new meaning to the term xe2x80x9cweed controlxe2x80x9d; for within this series of compounds it has been found that some are broad-spectrum or total vegetation herbicides with activity in both herbaceous and woody plants while others are highly selective weed control agents useful in the presence of crops. The imidazolinones are best exemplified by the commercial compounds imazethapyr, imazaquin, imazametha-benz-methyl and imazapyr.
Imidazolinone compounds are used by farmers throughout the world to control undesirable plant species. In certain areas, several imidazolinone compounds are individually used to control weeds growing in the presence of different crops such as soybeans, corn, peanuts, wheat, barley, rye and sunflowers, among others.
Although imidazolinone compounds gradually degrade in the environment, residues remaining in the soil should be monitored so as to provide the most efficacious weed control while avoiding possible damage to fallow crop plants. It is particularly important to monitor individual imidazolinone residues in those areas where several imidazolinones are used.
An enzyme-linked immunosorbent assay for the detection of imidazolinone compounds is described in U.S. Pat. No. 5,342,771. However, the polyclonal antibodies described in that patent application are not specific enough to determine the presence and concentration of an imidazolinone compound in the presence of one or more other imidazolinone compounds.
It is an object of the present invention to provide haptens and antigens and enzyme conjugates prepared therefrom which are useful in a method for determining the presence and concentration of an imidazolinone compound in the presence of one or more other imidazolinone compounds.
The present invention provides imidazolinone haptens and antigens and enzyme conjugates which are prepared from the imidazolinone haptens.
The antigens of the present invention are used to prepare imidazolinone specific monoclonal antibodies. Advantageously, certain of those monoclonal antibodies are used to determine the presence and concentration of an imidazolinone compound in the presence of one or more other imidazolinone compounds.
The enzyme conjugates of the present invention are used in a direct competitive immunoassay to determine the presence and concentration of an imidazolinone compound.
The present invention provides imidazolinone haptens that are used to prepare antigens which elicit imidazolinone specific antibody production in animals.
The imidazolinone haptens of the present invention have the structural formula 
wherein
R is hydrogen or C1-C4alkyl;
R1 is C1-C4alkyl;
R2 is C1-C4alkyl or C3-C6cycloalkyl; and when R1 and R2 are taken together with the carbon atom to which they are attached they may represent C3-C6cycloalkyl optionally substituted with methyl;
Y and Z are each independently hydrogen, halogen, C1-C6-alkyl, C1-C4alkoxyalkyl, C1-C4hydroxyalkyl, C1-C4alkoxy, C1-C4alkylthio, phenoxy, C1-C4haloalkyl, nitro, cyano, C1-C4alkylamino, di(C1-C4alkyl)amino, C1-C4alkylsulfonyl or phenyl optionally substituted with one C1-C4alkyl group, C1-C4alkoxy group or halogen atom; and when taken together, Y and Z may form a ring in which YZ are represented by the structure: xe2x80x94(CH2)nxe2x80x94, where n is an integer of 3 or 4; or 
where R3, R4, R5 and R6 are each independently hydrogen, halogen, C1-C4alkyl or C1-C4alkoxy;
A is N or CH;
W is
C3-C12alkylene optionally substituted with one or two C1-C3alkyl groups, C1-C3alkoxy groups, oxo groups or halogen atoms, and optionally interrupted by one oxygen or sulfur atom,
C3-C12alkenylene optionally substituted with one or two C1-C3alkyl groups, C1-C3alkoxy groups, oxo groups or halogen atoms, and optionally interrupted by one oxygen or sulfur atom, or
C3-C12alkynylene optionally substituted with one or two C1-C3alkyl groups, C1-C3alkoxy groups, oxo groups or halogen atoms, and optionally interrupted by one oxygen or sulfur atom; and
W1 is
C1-C12alkylene optionally substituted with one or two C1-C3alkyl groups, C1-C3alkoxy groups, oxo groups or halogen atoms, and optionally interrupted by one oxygen or sulfur atom,
C2-C12alkenylene optionally substituted with one or two Cl-C3alkyl groups, C1-C3alkoxy groups, oxo groups or halogen atoms, and optionally interrupted by one oxygen or sulfur atom, or
C3-C12alkynylene optionally substituted with one or two C1-C3alkyl groups, C1-C3alkoxy groups, oxo groups or halogen atoms, and optionally interrupted by one oxygen or sulfur atom.
Preferred formula I and II haptens are those wherein
R is hydrogen or methyl;
R1 is methyl;
R2 is isopropyl;
Y is hydrogen, C1-C4alkyl or C1-C4alkoxyalkyl;
Z is hydrogen; and when taken together, Y and Z may form a ring in which YZ is represented by the structure: xe2x80x94CHxe2x95x90CHxe2x80x94CHxe2x95x90CHxe2x80x94;
A is N or CH;
W is C3-C12alkylene or C3-C12alkenylene; and
W1 is C1-C12alkylene or C2-C12alkenylene.
More preferred formula I and II haptens which are used to prepare antigens which elicit imidazolinone specific antibody production in animals are those wherein
R is hydrogen or methyl;
R1 is methyl;
R2 is isopropyl;
Y is hydrogen, methyl, ethyl or methoxymethyl;
Z is hydrogen; and when taken together, Y and Z may form a ring in which YZ is represented by the structure: xe2x80x94CHxe2x95x90CHxe2x80x94CHxe2x95x90CHxe2x80x94;
A is N;
W is C3-C6alkylene or C3-C6alkenylene; and
W1 is C1-C6alkylene or C2-C6alkenylene.
Imidazolinone haptens of the present invention which are particularly useful in the preparation of antigens include
6-[5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)nicotinamido]hexanoic acid;
2-(5-ethyl-2-pyridyl)-4-isopropyl-4-methyl-5-oxo-2-imidazoline-1-valeric acid;
4-[5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)nicotinamido]butyric acid;
2-(5-ethyl-2-pyridyl)-4-isopropyl-4-methyl-5-oxo-2-imidazoline-1-crotonic acid, (E)-;
2-(5-ethyl-2-pyridyl)-4-isopropyl-4-methyl-5-oxo-2-imidazoline-1-acetic acid; and
4-[5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-N-methylnicotinoamido]butyric acid,
among others.
The antigens of the present invention which are prepared from the imidazolinone haptens described above and are useful in the production of imidazolinone specific antibodies in animals have the structural formula 
wherein
R is hydrogen or C1-C4alkyl;
R1 is C1-C4alkyl;
R2 is C1-C4alkyl or C3-C6cycloalkyl; and when R1 and R2 are taken together with the carbon atom to which they are attached they may represent C3-C6cycloalkyl optionally substituted with methyl;
Y and Z are each independently hydrogen, halogen, C1-C6-alkyl, C1-C4alkoxyalkyl, Cl-C4hydroxyalkyl, C1-C4alkoxy, C1-C4alkylthio, phenoxy, C1-C4haloalkyl, nitro, cyano, C1-C4alkylamino, di(C1-C4alkyl)amino, C1-C4alkylsulfonyl or phenyl optionally substituted with one C1-C4alkyl group, C1-C4alkoxy group or halogen atom; and when taken together, Y and Z may form a ring in which YZ are represented by the structure: xe2x80x94(CH2)nxe2x80x94, where n is an integer of 3 or 4; or 
where R3, R4, R5 and R6 are each independently hydrogen, halogen, C1-C4alkyl or C1-C4alkoxy;
A is N or CH;
W is
C3-C12alkylene optionally substituted with one or two C1-C3alkyl groups, C1-C3alkoxy groups, oxo groups or halogen atoms, and optionally interrupted by one oxygen or sulfur atom,
C3-C12alkenylene optionally substituted with one or two C1-C3alkyl groups, C1-C3alkoxy groups, oxo groups or halogen atoms, and optionally interrupted by one oxygen or sulfur atom, or
C3-C12alkynylene optionally substituted with one or two C1-C3alkyl groups, C1-C3alkoxy groups, oxo groups or halogen atoms, and optionally interrupted by one oxygen or sulfur atom;
W1 is
C1-C12alkylene optionally substituted with one or two C1-C3alkyl groups, C1-C3alkoxy groups, oxo groups or halogen atoms, and optionally interrupted by one oxygen or sulfur atom,
C2-C12alkenylene optionally substituted with one or two C1-C3alkyl groups, C1-C3alkoxy groups, oxo groups or halogen atoms, and optionally interrupted by one oxygen or sulfur atom, or
C3-C12alkynylene optionally substituted with one or two Cl-C3alkyl groups, Cl-C3alkoxy groups, oxo groups or halogen atoms, and optionally interrupted by one oxygen or sulfur atom;
X is an integer from about 1 to 40; and
Q is a protein.
Proteins suitable for use in the antigens of this invention include cationized bovine serum albumin, keyhole limpet hemocyanin, ovalbumin, bovine serum albumin, thyroglobulin, human serum albumin, fibrinogen and the like.
Preferred formula III and IV antigens of the present invention are those wherein
R is hydrogen or methyl;
R1 is methyl;
R2 is isopropyl;
Y is hydrogen, C1-C4alkyl or C1-C4alkoxyalkyl;
Z is hydrogen; and when taken together, Y and Z may form a ring in which YZ is represented by the structure: xe2x80x94CHxe2x95x90CHxe2x80x94CHxe2x95x90CHxe2x80x94;
A is N or CH;
W is C3-C12alkylene or C3-C12alkenylene;
W1 is C1-C12alkylene or C2-C12alkenylene;
X is an integer from about 1 to 40; and
Q is a protein.
More preferred formula III and IV antigens which elicit imidazolinone specific antibody production in animals are those wherein
R is hydrogen or methyl;
R1 is methyl;
R2 is isopropyl;
Y is hydrogen, methyl, ethyl or methoxymethyl;
Z is hydrogen; and when taken together, Y and Z may form a ring in which YZ is represented by the structure: xe2x80x94CHxe2x95x90CHxe2x80x94CHxe2x95x90CHxe2x80x94;
A is N;
W is C3-C6alkylene or C3-C6alkenylene;
W1 is C1-C6alkylene or C2-C6alkenylene;
X is an integer from about 1 to 40; and
Q is a protein.
Antigens of the present invention which are especially useful in the production of imidazolinone specific antibodies are those having the following structural formula 
wherein
X is an integer from about 1 to 40;
OVA is ovalbumin; and
Q is keyhole limpet hemocyanin or cationized bovine serum albumin.
The present invention further provides enzyme conjugates which are used in a direct competition immunoassay to determine the presence of imidazolinone compounds in water, soil or plant samples.
The enzyme conjugates of the present invention have the structural formula 
wherein
R is hydrogen or Cl-C4alkyl;
R1 is Cl-C4alkyl;
R2 is Cl-C4alkyl or C3-C6cycloalkyl; and when R1 and R2 are taken together with the carbon atom to which they are attached they may represent C3-C6cycloalkyl optionally substituted with methyl;
Y and Z are each independently hydrogen, halogen, C1-C6-alkyl, C1-C4alkoxyalkyl, C1-C4hydroxyalkyl, C1-C4alkoxy, C1-C4alkylthio, phenoxy, C1-C4haloalkyl, nitro, cyano, C1-C4alkylamino, di(C1-C4alkyl)amino, C1-C4alkylsulfonyl or phenyl optionally substituted with one C1-C4alkyl group, C1-C4alkoxy group or halogen atom; and when taken together, Y and Z may form a ring in which YZ are represented by the structure: xe2x80x94(CH2)nxe2x80x94, where n is an integer of 3 or 4; or 
where R3, R4, R5 and R6 are each independently hydrogen, halogen, Cl-C4alkyl or C1-C4alkoxy;
A is N or CH;
W is
C3-C12alkylene optionally substituted with one or two C1-C3alkyl groups, C1-C3alkoxy groups, oxo groups or halogen atoms, and optionally interrupted by one oxygen or sulfur atom,
C3-C12alkenylene optionally substituted with one or two C1-C3alkyl groups, C1-C3alkoxy groups, oxo groups or halogen atoms, and optionally interrupted by one oxygen or sulfur atom, or
C3-C12alkynylene optionally substituted with one or two C1-C3alkyl groups, C1-C3alkoxy groups, oxo groups or halogen atoms, and optionally interrupted by one oxygen or sulfur atom;
W1 is
C1-C12alkylene optionally substituted with one or two C1-C3alkyl groups, C1-C3alkoxy groups, oxo groups or halogen atoms, and optionally interrupted by one oxygen or sulfur atom,
C2-C12alkenylene optionally substituted with one or two Cl-C3alkyl groups, C1-C3alkoxy groups, oxo groups or halogen atoms, and optionally interrupted by one oxygen or sulfur atom, or
C3-C12alkynylene optionally substituted with one or two C1-C3alkyl groups, C1-C3alkoxy groups, oxo groups or halogen atoms, and optionally interrupted by one oxygen or sulfur atom; and
E is an enzyme.
Enzymes suitable for use in the enzyme conjugates of the present invention include those that produce a color change when treated with a substrate. Preferred enzymes are alkaline phosphatase, horseradish peroxidase, urease, glucose oxidase and galactosidase.
Preferred formula VIII and IX enzyme conjugates are those wherein
R is hydrogen or methyl;
R1 is methyl;
R2 is isopropyl;
Y is hydrogen, Cl-C4alkyl or C1-C4alkoxyalkyl;
Z is hydrogen; and when taken together, Y and Z may form a ring in which YZ is represented by the structure: xe2x80x94CHxe2x95x90CHxe2x80x94CHxe2x95x90CHxe2x80x94;
A is N or CH;
W is C3-C12alkylene or C3-C12alkenylene;
W1 is C1-C12alkylene or C2-C12alkenylene; and
E is an enzyme.
More preferred formula VIII and IX enzyme conjugates of the present invention are those wherein
R is hydrogen or methyl;
R1 is methyl;
R2 is isopropyl;
Y is hydrogen, methyl, ethyl or methoxymethyl;
Z is hydrogen; and when taken together, Y and Z may form a ring in which YZ is represented by the structure: xe2x80x94CHxe2x95x90CHxe2x80x94CHxe2x95x90CHxe2x80x94;
A is N;
W is C3-C6alkylene or C3-C6alkenylene;
W1 is C1-C6alkylene or C2-C6alkenylene; and
E is an enzyme.
One of the most preferred enzyme conjugates of this invention has the following structural formula 
wherein
E is alkaline phosphatase.
The formula I haptens of the present invention may be prepared by reacting a compound having the structural formula XI 
wherein Y, Z, A, R1 and R2 are as described hereinabove with an amino acid compound having the structural formula XII 
wherein R and W are as described hereinabove; and the acid addition salts thereof in the presence of a trialkylamine such as triethylamine and a polar solvent such as N,N-dimethylformamide to form a reaction mixture which is quenched with aqueous acid to form the desired formula I hapten. The above reaction scheme is shown in Flow Diagram I. 
Alternatively, formula I haptens may be prepared by reacting a compound having the structural formula XI with an amino acid ester having the structural formula XIII 
wherein
R and W are as described hereinabove;
R7 is C1-C4alkyl; and
the acid addition salts thereof
in the presence of a trialkylamine such as triethylamine and a polar solvent such as N,N-dimethylformamide to form a reaction mixture which is quenched with aqueous acid to obtain an ester compound having the structural formula XIV 
wherein Y, Z, W, R1, R2 and R7 are as described hereinabove, saponifying the formula XIV ester compound with aqueous base and quenching the saponification reaction mixture with acid to form the desired formula I hapten. The reaction scheme is shown in Flow Diagram II. 
Formula II haptens may be prepared by reacting a compound having the structural formula XV 
wherein Y, Z, A, R1 and R2 are as described hereinabove with a metal hydride such as sodium hydride and a halo ester compound having the structural formula XVI 
wherein W1 and R7 are as described hereinabove; and X1 is halogen to form a compound having the structural formula XVII 
wherein Y, Z, W1, R1, R2 and R7 are as described hereinabove, saponifying the formula XVII compound with aqueous base and quenching the saponification reaction mixture with acid to obtain the desired formula II hapten. The reaction scheme is shown in Flow Diagram III. 
The antigens of the present invention may be prepared by reacting a hapten of the present invention with at least about one molar equivalent of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to form an activated hapten and reacting the activated hapten with the amino groups of a protein to form the desired antigen. The above reaction scheme is shown in Flow Diagrams IV(a) and IV(b). 
Similarly, the enzyme conjugates of the present invention may be prepared as shown below in Flow Diagrams V(a) and V(b). 
The antigens of the present invention are used to elicit imidazolinone specific antibody production in animals. Several weeks after an animal is immunized with one or more antigens, the serum is harvested and the polyclonal antibodies present in the serum may be used in indirect or direct immunoassays to determine the presence and quantity of imidazolinone compounds in soil, water or plant samples.
Advantageously, it has been found that certain hybridomas, prepared by fusing a spleen cell from an animal immunized with an antigen of the present invention with a myeloma cell, produce monoclonal antibodies which are specific for one imidazolinone compound among others. Heretofore, imidazolinone antibodies were not specific enough to allow for the accurate determination of the presence and quantity of an imidazolinone compound in the presence of one or more other imidazolinone compounds. The methods used to prepare the polyclonal antibodies, hybridomas and monoclonal antibodies of the present invention are described in the examples. Also described in the examples are the indirect and direct immunoassay methods which are used to determine the presence and quantity of an imidazolinone compound present in a sample.
The imidazolinone compounds are represented by structural formula XVIII 
wherein
Y, Z, A, R1 and R2 are as described hereinabove; and
R8 is hydrogen or C1-C4alkyl.
The polyclonal and monoclonal antibodies of this invention are especially useful in indirect and direct immunoassays to determine the presence and quantity of the following imidazolinone compounds:
5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)nicotinic acid (imazethapyr);
2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-3-quinolinecarboxylic acid (imazaquin);
mixture of methyl 6-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-m-toluate and methyl 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-p-toluate (imazamethabenz-methyl);
2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)nicotinic acid (imazapyr); and
2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-methylnicotinic acid (imazamethapyr).
Representative hybridoma cell lines of the present invention have been deposited on Aug. 26, 1999 pursuant to the Budapest Treaty for purposes of patent procedure and accepted by the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209 U.S.A. The deposits of the mouse hybridoma cell lines, which are identified as 1D2.6, 2C6.5, 1A5.5, 3A5.1 and 3A2.2, have been assigned the patent deposit designation numbers PTA-579, PTA-580, PTA-581, PTA-582 and PTA-583, respectively.